James H. Orf

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

Targeted comparative genome analysis and qualitative mapping of a major partial-resistance gene to the soybean cyst nematode.

A major partial-resistance locus to the soybean cyst nematode (Heterodera glycines Ichinohe; SCN) was identified on linkage group ‘G’ of soybean [Glycine max (L.) Merr.] using restriction fragment length polymorphisms (RFLPs). This locus explained 51.4% (LOD=10.35) of the total phenotypic variation in disease response in soybean Plant Introduction (PI) 209332, 52.7% (LOD=15.58) in PI 90763, 40.0% (LOD=10.50) in PI 88788, and 28.1% (LOD=6.94) in ‘Peking’. Initially, the region around this major resistance locus was poorly populated with DNA markers. To increase marker density in this genomic region, first random, and later targeted, comparative mapping with RFLPs from mungbean [Vigna radiata (L.) R. Wilcz.] and common bean (Phaseolus vulgaris L.) was performed, eventually leading to one RFLP marker every 2.6 centimorgans (cM). Even with this marker density, the inability to resolve SCN disease response into discrete Mendelian categories posed a major limitation to mapping. Thus, qualitative scoring of SCN disease response was carried out in an F5∶6 recombinant inbred population derived from ‘Evans’xPI 209332 using a 30% disease index cut-off for resistance. Using the computer program JoinMap, an integrated map of the region of interest was created, placing the SCN resistance locus 4.6 cM from RFLP marker B53 and 2.8 cM from Bng30. This study demonstrates how a combination of molecularmapping strategies, including comparative genome analysis, join mapping, and qualitative scoring of a quantitative trait, potentially provide the necessary tools for high-resolution mapping around a quantitative-trait locus.

Soybean Cyst Nematode Population Development and Associated Soybean Yields of Resistant and Susceptible Cultivars in Minnesota

The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean yield-limiting factor, and the use of resistant cultivars is one of the most effective means to manage the nematode. During the past decade, a number of resistant cultivars in maturity groups I and II have been developed and made available to growers. A total of 47 resistant cultivars and nine susceptible cultivars were evaluated at 15 SCN-infested field sites and two noninfested sites during 1996 to 1998 in Minnesota. As expected, more nematodes developed on susceptible cultivars than on resistant cultivars. Egg density on susceptible cultivars increased by 1.9- to 10.6-fold during the growing season at 12 sites and did not change at the other three sites. Average egg density decreased over time for resistant cultivars at all sites, except where the initial egg density was low (≤455 eggs per 100 cm3 soil). Nematode reproduction factors (Rf = egg density at harvest/egg density at planting) for individual resistant and susceptible cultivars were highly consistent across the eight sites where initial SCN density was more than 1,000 eggs per 100 cm3 soil. Resistance, however, varied among the cultivars, with the average Rf of individual resistant cultivars across the sites ranging from 0.3 to 1.7. Resistant cultivars produced an average yield of 3,082 kg/ha compared with 2,497 kg/ha by susceptible cultivars at eight of 10 sites where egg density at planting was greater than 700 eggs per 100 cm3 soil. In contrast, no difference in yield was observed between resistant and susceptible cultivars at sites where egg density at planting was lower than 500 eggs per 100 cm3 soil. Yield differences between resistant and susceptible cultivars increased with increasing initial SCN egg density. In six fields infested with initial densities of more than 5,000 eggs per 100 cm3 soil, resistant cultivars produced 28.4% (676 kg/ha) more yield on average than susceptible cultivars. Soybean yield increased when cultivars with increasing resistance to the SCN (lower Rf or females formed on roots) were grown in fields infested with SCN. Average relative yield (yield of a cultivar/average yield of all resistant cultivars at a site) of individual resistant cultivars across all SCN-infested sites ranged from 0.76 to 1.10. Yield consistency of soybean cultivars was low among the different sites, indicating that many other factors affected yield. Our results suggest growing resistant cultivars is an effective method to manage SCN in Minnesota while minimizing yield loss due to SCN.

Determining the linkage of quantitative trait loci to RFLP markers using extreme phenotypes of recombinant inbreds of soybean (Glycine max L. Merr.)

An experimental test is described for linkages between RFLP markers and quantitative trait loci (QTL). Two hundred and eighty-four F7-derived recombinant inbred lines (RIL) obtained from crossing the soybean cultivars (Glycine max L. Merr.) ‘Minsoy’ and ‘Noir 1’ were evaluated for maturity, plant height, lodging, and seed yield. RIL exhibiting an extreme phenotype for each trait (earliest and latest plants for maturity, etc.) were selected, and two bulked DNA samples were prepared for each trait. A Southern transfer of the digested bulked DNA was hybridized with restriction fragement length polymorphism (RFLP) probes, and linkages with QTL were established by quantitating the amount of radioactive probe that bound to fragments defining alternative parental RFLP alleles. When an RFLP marker was linked to a QTL, one parental allele predominated in the bulked DNA from a particular phenotype; the other allele was associated with the opposite phenotype. When linkage was absent, radioactivity was associated equally with both alleles for a given phenotype (or with both phenotypes for a given allele). These results confirmed RFLP-QTL associations previously discovered by interval mapping on a smaller segregating population from the same cross. New linkages to QTL were also verified.

Increasing Crop Competitiveness to Weeds Through Crop Breeding

Abstract Increasing the ability of crops to compete against weeds, through either enhancing crop tolerance or crop interference to weeds, provides an attractive addition to current weed control practices and could be an integral component of weed management systems. Research has shown that considerable variability exists among crop culti-vars with respect to their ability to compete with weeds. Despite this evidence, directed research on competitive crops has been minimal. Reasons for this lack of emphasis in plant breeding programs include the effectiveness of current weed management with tillage and herbicides, and the lack of easily identifiable crop characteristics that are indicative of weed competitiveness. Expanded knowledge of specific crop-weed interactions would facilitate crop competitiveness to weeds through either crop management practices or plant breeding. Plant breeders need basic and applied information to identify favorable crop-weed competitive traits in order to enhance or incorporate ...

Epistatic expression of quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] determined by QTL association with RFLP alleles.

Quantitative trait values for seed oil and protein content or for maturity were measured in recombinant inbred lines (RIL) of soybean derived from a cross between two soybean cultivars: ‘Minsoy’ PI 27890 and ‘Noir 1’ PI 290136. Seed oil was found to be inversely correlated to protein content. By analyzing DNA from plants with extreme phenotypes, we were able to identify quantitative trait loci (QTL) for these traits as being linked to several restriction fragment length polymorphism (RFLP) loci, including R183 for oil and protein content and R79 for maturity. Cumulative distributions of trait values were graphed for those RIL with ‘Minsoy’ alleles and for those with ‘Noir 1’ alleles. As already suggested by the alleles found associated with extreme phenotypes, the distributions were consistent with an independent and additive expression of the maturity QTL linked to R79. That is, the cumulative distributions for plants with ‘Minsoy’ alleles and for plants with ‘Noir 1’ alleles were similar in shape, but the entire ‘Noir 1’ curve had been shifted to later maturity dates. In contrast, the trait distributions for a locus affecting oil and protein content linked to R183 were not compatible with an additive model. These results suggest that this approach can be used for rapid identification of QTLs with epistatic expression.

Root iron‐reduction capacity for genotypic evaluation of iron efficiency in soybean

Abstract Genetic resistance to Fe‐deficiency chlorosis is the most viable and economical means to overcome this problem in soybean [Glycine max (L.) Merr.], but current field evaluation is slowed and constrained by soil heterogeneity and environmental fluctuation. Highly resistant (Fe‐efficient) cultivars have been shown to reduce Fe3+ to Fe2+ more actively by the roots under Fe‐deficiency stress than highly susceptible genotypes. The objective of this study was to determine if Fe3+ reduction could be used to predict the degree of resistance or susceptibility to Fe‐deficiency chlorosis. Thirteen genotypes (both commercial and experimental) with known field susceptibility ratings were grown in a growth chamber in modified Hoagland solution. The more Fe‐efficient genotypes reduced Fe3+ earlier and to a greater extent than the less Fe‐efficient types. The sum of the seven daily Fe3+ reduction measurements was negatively correlated with field chlorosis ratings, as high as ‐0.864 (p > 0.01), and was a good pre...

Potential of Association Mapping and Genomic Selection to Explore PI 88788 Derived Soybean Cyst Nematode Resistance

The potential of association mapping (AM) and genomic selection (GS) has not yet been explored for investigating resistance to soybean cyst nematode (SCN), the most destructive pest affecting soybean. We genotyped 282 representative accessions from the University of Minnesota soybean breeding program using a genome‐wide panel of 1536 single nucleotide polymorphism (SNP) markers and evaluated plant responses to SCN HG type 0. After adjusting for population structure, AM detected significant signals at two loci corresponding to rhg1 and FGAM1 plus a third locus located at the opposite end of chromosome 18. Our analysis also identified a discontinuous long‐range haplotype of over 600 kb around rhg1 locus associated with resistance to SCN HG type 0. The same phenotypic and genotypic datasets were then used to access GS accuracy for prediction of SCN resistance in the presence of major genes through a sixfold cross‐validation study. Genomic selection using the full marker set produced average prediction accuracy ranging from 0.59 to 0.67 for SCN resistance, significantly more accurate than marker‐assisted selection (MAS) strategies using two rhg1‐associated DNA makers. Reducing the number of markers to 288 SNPs in the GS training population had little effect on genomic prediction accuracy. This study demonstrates that AM can be an effective genomic tool for identifying genes of interest in diverse germplasm. The results also indicate that improved MAS and GS can enhance breeding efficiency for SCN resistance in existing soybean improvement programs.

Genetic variance, coefficient of parentage, and genetic distance of six soybean populations

Plant breeders would like to predict which biparental populations will have the largest genetic variance. If the population genetic variance could be predicted using coefficient of parentage or genetic distance estimates based on molecular marker data, breeders could choose parents that produced segregating populations with a large genetic variance. Three biparental soybean Glycine max (L.) Merr. populations were developed by crossing parents that were closely related, based on pedigree relationships. Three additional biparental populations were developed by crossing parents that were assumed to be unrelated. The genetic variance of each population was estimated for yield, lodging, physiological maturity, and plant height. Coefficient of parentage was calculated for each pair of parents used to develop the segregating populations. Genetic distance was determined, based on the number of random amplified polymorphic markers (RAPD) that were polymorphic for each pair of parents. Genetic distance was not associated with the coefficient of parentage or the magnitude of the genetic variance. The genetic variance pooled across the three closely related populations was smaller than the genetic variance pooled across the three populations derived from crossing unrelated parents for all four traits that were evaluated.

Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the phenotypes of MinnGold and T219H are clearly distinct, sequencing of ChlI1a in T219H identified a different nonsynonymous mutation in the third exon, only six base pairs from the MinnGold mutation. This information, along with previously published allelic tests, were used to identify and clone a third yellow foliage mutation, CD-5, which was previously mapped to chromosome 15. This mutation was identified in the ChlI1b gene, a paralog of ChlI1a. Sequencing of the ChlI1b allele in CD-5 identified a nonsynonymous substitution in the third exon that confers an identical amino acid change as the T219H substitution at ChlI1a. Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species. These results suggest that amino acid alterations in this critical domain may create competitive inhibitory interactions between the mutant and wild-type ChlI1a and ChlI1b proteins.